The long term goal of this project is to identify the macromolecular interactions required for transcription of the yeast U6 small nuclear RNA gene by RNA polymerase m (RNAP III). Recognition of genes by RNAP III requires the prior binding of specific factors to short promoter elements, resulting in formation of a stable pre-initiation complex. Initiation, elongation and termination of transcription then ensue. Transcript release and recycling of polymerase may require the action of additional factors. The yeast U6 RNA gene, SNR6, has an unusual promoter structure that we have exploited to study the role of the promoter elements and their associated factors in transcription initiation. A major focus is the B block promoter element, which binds the transcription initiation factor TFIIIC. First identified as a intragenic promoter element in tRNA genes, the SNR6 B block is in a unprecedented location, more than 100 base pairs downstream of the coding region. In addition to raising interesting questions concerning the structure of the pre-initiation complex, the extragenic location of the SNR6 B block provides a unique opportunity to subject this element to detailed genetic analysis. A specific aim of this study is to identify essential base pairs in the B block promoter element, and to determine which amino acids in the B block binding polypeptide (the 145 kD subunit of TFIIIC) contact these base pairs. We are also interested in the interaction of TFIIIB, the transcription factor that positions RNAP III, with promoter elements flanking the initiation site. The yeast U6 gene has both an A block element downstream, and a TATA box element upstream, of the start site. The A block is known to determine start site selection in tRNA genes, but we have found that the SNR6 TATA box also influences the position of transcription initiation. This finding is particularly intriguing because TFIIIB has recently been shown to contain the TFIID TATA-binding protein (TBP) as a subunit A second specific aim of this study is to determine how sequences flanking the SNR6 start site direct RNAP III to initiate transcription at the correct position. The mechanism of transcript release and recycling of RNAP III is poorly understood. Two factors have been implicated in this process: the human autoantigen La and TFIIIR, a silk worm transcription factor composed entirely of RNA. The precise role of these factors, or indeed their direct involvement in transcription in general has not been firmly established. A third specific aim of this study is to identify the yeast homologs of the La protein and TFIIIR, and to examine the role of these putative recycling factors in transcription of the yeast U6 RNA gene and other yeast RNAP III transcription units. By exploiting the unique properties of the U6 RNA gene, and the powerful genetic methodology available in the yeast model system, we will gain a better understanding of the mechanism of the first step of eukaryotic gene expression.